Thus it is known to place a cell suspension in a generally tubular sample chamber having an open end juxtaposed to the surface of a microscope or like slide, with the interposition of an apertured filter card that both provides a seal between the sample chamber and the slide surface and also serves to absorb the liquid components of the suspension. The assembly of sample chamber, slide and filter card is subjected to centrifugation to cause the deposition of a layer of cells on the slide surface and the removal of the suspension liquid into the filter card. One example of cytocentrifugation apparatus especially adapted to perform this cell separation and deposition technique is disclosed in U.S. Pat. No. 4,391,710. For an earlier example of such apparatus, see also G-I-T FACHZEITSCHRIFT FUR DAS LABORATORIUM, Vol. 5, No. 1 (January 1971), Darmstadt DE, at page 51, left-hand column.
As hitherto practised, for instance with the centrifugation apparatus of U.S. Pat. No. 4,391,710, the sample chamber is sized to deposit cells on a rather small, generally circular, area of the slide and handles correspondingly small samples of cell suspension. The rather small areas of cell deposit thereby produced are adequate for microscopic examination by a suitably trained operative making a visual examination but it is recognised that examination of the deposit would be facilitated if the deposit were spread over a larger area of the slide and, ideally, as a monolayer of cells. The uniform deposition of cells in a monolayer over a substantial area would, for instance, greatly facilitate counting of cells per unit area as well as the recognition of particular cell types of interest. Moreover, recent proposals for the automated examination of cell deposits by optical scanning devices require, for reproducibility of results, cells to be deposited in a monolayer over a, preferably rectangular, relatively large area of a suitable receiving surface such as a glass slide.
It should be understood that when a small volume sample of a suspension of solids in a liquid and contained in a circular section tube is centrifuged to deposit its suspended solids over a circular slide area defined by a correspondingly sized aperture in a significantly larger filter card, the liquid of the suspension tends to flow symmetrically and radially of the deposit area to be uniformly absorbed in the surrounding filter card. This flow of the liquid tends to carry some of the suspended solids away from the centre of the deposit area and towards the margin thereof, to lead to variations in thickness in the deposited layer, this tending to be thicker at its periphery than at its centre. Nonetheless, by appropriate choices of filter card absorbency and dimensions of the sample chamber, and by restricting the volume of liquid in the suspension sample, it is generally possible to obtain a deposit that is useful for visual examination, at least over the majority of its total area.
When, however, an attempt is made to cause the deposition over a non-circular surface area and, especially, over a relatively large non-circular area of the receiving surface, using samples containing larger volumes of suspending liquid, the flow of liquid transversely of the receiving surface and into the filter card is no longer symmetrical and tends to cause significant thickness variations in the deposited layer, rendering much of the area thereof difficult to examine reliably by visual methods and substantially useless for examination by automated optical scanning techniques.
Ideally, to achieve a uniform deposit thickness over the whole area of the deposited layer, the arrangement should be such that, under the artificial gravitational field created by centrifugation, solids (e.g. cells) suspended in the sample are caused to deposit on the receiving surface before there is any significant flow of the suspending liquid transversely of that surface and into the surrounding filter card, to cause lateral displacement of suspended solids before these are deposited. Moreover, the flow of suspending liquid into the surrounding filter card, when it occurs, should be slow to minimise the risk of currents over the deposited solids disturbing these and altering their disposition on the surface.
The ideal sequence of solids deposition followed by liquid removal is essentially unattainable in practice, some flow of suspension liquid into the filter card during deposition of the suspended solids being inevitable. However, the deposition perturbations due to transverse liquid flow can, we have discovered, be mitigated by suitable restriction of the rate of flow of liquid to the filter card and by ensuring that the flow pattern is such that at any point on the periphery of the deposit area, the local liquid flow is normal to the periphery and occurs at a substantially constant velocity. By observing these criteria, any flow-induced migration of suspended solids towards the periphery of the deposit area will be essentially uniform so that unacceptable thickening of the deposited layer will be confined to a substantially uniform width marginal band that can be ignored without difficulty in subsequent visual inspection and that can also be readily ignored by an automated optical scanning of the deposit area.
In filter card such as is used in the centrifugation procedures of interest, it can be shown that the volumetric flow rate of a liquid by capillary action in the card material it is directly proportional to the cross-sectional area of the material in the flow path of the liquid. Accordingly the flow of liquid in a filter card from a particular region of the perimeter of an aperture therein can be restrained by restricting the flow cross-sectional area available in the card material for liquid flowing from that region of the aperture perimeter. This cross-sectional area restriction may be accomplished by physical removal of material, to form openings constituting liquid flow barriers therein as by cutting slots transverse to a liquid flow path to be restricted, by localised impregnation of the card with an occlusive, e.g. a hydrophobic, agent so as to inhibit liquid flow in the filter card region so impregnated, or by localised compression, as by clamping, of the card.